Involvement of extracellular vesicle microRNA clusters in developing healthy and Rett syndrome brain organoids.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression.

Heterogeneity of Lipid and Protein Cartilage Profiles Associated with Human Osteoarthritis with or without Type 2 Diabetes Mellitus.

Osteoarthritis (OA) is a multifactorial pathology and comprises a wide range of distinct phenotypes. In this context, the characterization of the different molecular profiles associated with each phenotype can improve the classification of OA. In particular, OA can coexist with type 2 diabetes mellitus (T2DM). This study investigates lipidomic and proteomic differences between human OA/T2DM- and OA/T2DM+ cartilage through a multimodal mass spectrometry approach. Human cartilage samples were obtained after total knee replacement from OA/T2DM- and OA/T2DM+ patients. Label-free proteomics was employed to study differences in protein abundance and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for spatially resolved-lipid analysis. Label-free proteomic analysis showed differences between OA/T2DM- and OA/T2DM+ phenotypes in several metabolic pathways such as lipid regulation. Interestingly, phospholipase A2 protein was found increased within the OA/T2DM+ cohort. In addition, MALDI-MSI experiments revealed that phosphatidylcholine and sphingomyelin species were characteristic of the OA/T2DM- group, whereas lysolipids were more characteristic of the OA/T2DM+ phenotype. The data also pointed out differences in phospholipid content between superficial and deep layers of the cartilage. Our study shows distinctively different lipid and protein profiles between OA/T2DM- and OA/T2DM+ human cartilage, demonstrating the importance of subclassification of the OA disease for better personalized treatments.

An in vitro model for hypertrophic adipocytes: Time-dependent adipocyte proteome and secretome changes under high glucose and high insulin conditions.

Obesity is the consequence of a positive energy balance and characterized by enlargement of the adipose tissue, which in part is due to hyperplasia and hypertrophy of the adipocytes. Not much is known about the transition of normal mature adipocytes to the hypertrophic state, which in vivo is very hard to study. Here, we have maintained mature human SGBS cells as a surrogate for adipocytes, changes of morphological and molecular metabolism of the adipocytes were monitored over the first 4 days and the last 4 days. In total, 393 cellular proteins and 246 secreted proteins were identified for further analysis. During the first 4 days of high glucose and insulin, the adipocytes seemed to prefer pyruvate as energy source, whereas beta-oxidation was down-regulated supporting lipid loading. Over time, lipid droplet fusion instead of lipid uptake became relatively important for growth of lipid droplets during the last 4 days. Moreover, ECM production shifted towards ECM turnover by the up-regulation of proteases over eight days. The present in vitro system provides insight into the metabolic changes of adipocytes under conditions of high glucose and insulin, which may help to understand the process of in vivo adipocyte hypertrophy during the development of obesity.

Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats.

Physical activity increases muscle protein synthesis rates. However, the impact of exercise on the coordinated up- and/or downregulation of individual protein synthesis rates in skeletal muscle tissue remains unclear. The authors assessed the impact of exercise on mixed muscle, myofibrillar, and mitochondrial protein synthesis rates as well as individual protein synthesis rates in vivo in rats. Adult Lewis rats either remained sedentary (n = 3) or had access to a running wheel (n = 3) for the last 2 weeks of a 3-week experimental period. Deuterated water was injected and subsequently administered in drinking water over the experimental period. Blood and soleus muscle were collected and used to assess bulk mixed muscle, myofibrillar, and mitochondrial protein synthesis rates using gas chromatography-mass spectrometry and individual muscle protein synthesis rates using liquid chromatography-mass spectrometry (i.e., dynamic proteomic profiling). Wheel running resulted in greater myofibrillar (3.94 ± 0.26 vs. 3.03 ± 0.15%/day; p < .01) and mitochondrial (4.64 ± 0.24 vs. 3.97 ± 0.26%/day; p < .05), but not mixed muscle (2.64 ± 0.96 vs. 2.38 ± 0.62%/day; p = .71) protein synthesis rates, when compared with the sedentary condition. Exercise impacted the synthesis rates of 80 proteins, with the difference from the sedentary condition ranging between -64% and +420%. Significantly greater synthesis rates were detected for F1-ATP synthase, ATP synthase subunit alpha, hemoglobin, myosin light chain-6, and synaptopodin-2 (p < .05). The skeletal muscle protein adaptive response to endurance-type exercise involves upregulation of mitochondrial protein synthesis rates, but it is highly coordinated as reflected by the up- and downregulation of various individual proteins across different bulk subcellular protein fractions.

Molecular adaptation in adipose tissue in response to overfeeding with a high-fat diet under sedentary conditions in South Asian and Caucasian men.

For the same BMI, South Asians have a higher body fat percentage than Caucasians. There might be differences in the fatty acid (FA) handling in adipose tissue when both ethnicities are exposed to high-fat overfeeding. The objective of the present study was to investigate the molecular adaptation in relation to FA metabolism in response to overfeeding with a high-fat diet (OHFD) in South Asian and Caucasian men. Ten South Asian men (BMI 18-29 kg/m2) and ten Caucasian men (BMI 22-33 kg/m2), matched for body fat percentage, aged 20-40 years were included. A weight-maintenance diet (30 % fat, 55 % carbohydrate and 15 % protein) was given for 3 d followed by 3 d of overfeeding (150 % energy requirement) with a high-fat diet (60 % fat, 25 % carbohydrate and 15 % protein) while staying in a respiration chamber. Before and after overfeeding, abdominal subcutaneous fat biopsies were taken. Proteins were isolated, analysed and quantified for short-chain 3-hydroxyacyl-CoA dehydrogenase (HADH), carnitine palmitoyl-transferase 1α (CPT1a), adipose TAG lipase, perilipin A (PLINA), perilipin B, lipoprotein lipase and fatty acid binding protein 4 using Western blotting. OHFD decreased the HADH level (P < 0·05) in Caucasians more than in Asians (P < 0·05), but the baseline and after intervention HADH level was relatively higher in Caucasians. The level of CPT1a decreased in South Asians and increased in Caucasians (P < 0·05). PLINA did not change with diet but the level was higher in South Asians (P < 0·05). The observed differences in HADH and PLINA levels as well as in CPT1a response may be important for differences in the long-term regulation of energy (fat) metabolism in these populations.

Brain tissue plasticity: protein synthesis rates of the human brain.

All tissues undergo continuous reconditioning via the complex orchestration of changes in tissue protein synthesis and breakdown rates. Skeletal muscle tissue has been well studied in this regard, and has been shown to turnover at a rate of 1-2% per day in vivo in humans. Few data are available on protein synthesis rates of other tissues. Because of obvious limitations with regard to brain tissue sampling no study has ever measured brain protein synthesis rates in vivo in humans. Here, we applied stable isotope methodology to directly assess protein synthesis rates in neocortex and hippocampus tissue of six patients undergoing temporal lobectomy for drug-resistant temporal lobe epilepsy (Clinical trial registration: NTR5147). Protein synthesis rates of neocortex and hippocampus tissue averaged 0.17 ± 0.01 and 0.13 ± 0.01%/h, respectively. Brain tissue protein synthesis rates were 3-4-fold higher than skeletal muscle tissue protein synthesis rates (0.05 ± 0.01%/h; P < 0.001). In conclusion, the protein turnover rate of the human brain is much higher than previously assumed.

Glucose Restriction Plus Refeeding in Vitro Induce Changes of the Human Adipocyte Secretome with an Impact on Complement Factors and Cathepsins.

Adipose tissue is a major endocrine organ capable of secreting adipokines with a role in whole-body metabolism. Changes in the secretome profile during the development of obesity is suspected to contribute to the risk of health complications such as those associated with weight regain after weight loss. However, the number of studies on weight regain is limited and secretome changes during weight regain have hardly been investigated. In an attempt to generate leads for in vivo studies, we have subjected human Simpson Golabi Behmel Syndrome adipocytes to glucose restriction (GR) followed by refeeding (RF) as an in vitro surrogate for weight regain after weight loss. Using LC-MS/MS, we compared the secreted protein profile after GR plus RF with that of normal feeding (NF) to assess the consequences of GR plus RF. We identified 338 secreted proteins of which 49 were described for the first time as being secreted by adipocytes. In addition, comparison between NF and GR plus RF showed 39 differentially secreted proteins. Functional classification revealed GR plus RF-induced changes of enzymes for extracellular matrix modification, complement system factors, cathepsins, and several proteins related to Alzheimer's disease. These observations can be used as clues to investigate metabolic consequences of weight regain, weight cycling or intermittent fasting.

Identification and Characterization of Cardiac Troponin T Fragments in Serum of Patients Suffering from Acute Myocardial Infarction.

BACKGROUND: Cardiac troponin T (cTnT) is the preferred biomarker for the diagnosis of acute myocardial infarction (AMI). It has been suggested that cTnT is present predominantly in fragmented forms in human serum following AMI. In this study, we have used a targeted mass spectrometry assay and epitope mapping using Western blotting to confirm this hypothesis. METHODS: cTnT was captured from the serum of 12 patients diagnosed with AMI using an immunoprecipitation technique employing the M11.7 catcher antibody and fractionated with SDS-PAGE. Coomassie-stained bands of 4 patients at 37, 29, and 16 kDa were excised from the gel, digested with trypsin, and analyzed on a Q Exactive instrument set on targeted Selected Ion Monitoring mode with data-dependent tandem mass spectrometry (MS/MS) for identification. Western blotting employing 3 different antibodies was used for epitope mapping. RESULTS: Ten cTnT peptides of interest were targeted. By using MS/MS, all of these peptides were identified in the 37-kDa, intact, cTnT band. In the 29- and 16-kDa fragment bands, 8 and 4 cTnT-specific peptides were identified, respectively. Some of these peptides were "semitryptic," meaning that their C-termini were not formed by trypsin cleavage. The C-termini of these semitryptic peptides represent the C-terminal end of the cTnT molecules present in these bands. These results were confirmed independently by epitope mapping. CONCLUSIONS: Using LC-MS, we have succeeded in positively identifying the 29- and 16-kDa fragment bands as cTnT-derived products. The amino acid sequences of the 29- and 16-kDa fragments are Ser79-Trp297 and Ser79-Gln199, respectively.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Weight loss-induced stress in subcutaneous adipose tissue is related to weight regain.

Initial successful weight loss is often followed by weight regain after the dietary intervention. Compared with lean people, cellular stress in adipose tissue is increased in obese subjects. However, the relation between cellular stress and the risk for weight regain after weight loss is unclear. Therefore, we determined the expression levels of stress proteins during weight loss and weight maintenance in relation to weight regain. In vivo findings were compared with results from in vitro cultured human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. In total, eighteen healthy subjects underwent an 8-week diet programme with a 10-month follow-up. Participants were categorised as weight maintainers or weight regainers (WR) depending on their weight changes during the intervention. Abdominal subcutaneous adipose tissue biopsies were obtained before and after the diet and after the follow-up. In vitro differentiated SGBS adipocytes were starved for 96 h with low (0·55 mm) glucose. Levels of stress proteins were determined by Western blotting. WR showed increased expressions of ß-actin, calnexin, heat shock protein (HSP) 27, HSP60 and HSP70. Changes of ß-actin, HSP27 and HSP70 are linked to HSP60, a proposed key factor in weight regain after weight loss. SGBS adipocytes showed increased levels of ß-actin and HSP60 after 96 h of glucose restriction. The increased level of cellular stress proteins in the adipose tissue of WR probably resides in the adipocytes as shown by in vitro experiments. Cellular stress accumulated in adipose tissue during weight loss may be a risk factor for weight regain.